Citations

AbstractSeveral studies in the past have formed 3‐dimensional (3D) spheroids of primary hepatocytes in suspension culture. Unfortunately, primary hepatocytes in a suspension environment tend to lose their differentiated function over time, generally due to damage from fluid shear stress and eventual spheroid settling. We have therefore created a novel suspension culture system, by seeding H35 rat hepatoma cells, a hepatocyte‐derived cell line, in a 24‐well tissue culture polystyrene (TCPS) plate placed atop an orbital shaker to create 3D spheroids. To provide stability to the formed spheroids, we used a long‐chain polymer, bovine serum albumin (BSA), dissolved in the cell culture medium and/or coated on TCPS surfaces placed in suspension configurations. Our results demonstrate that BSA coating of culture surfaces resulted in uniform and well‐defined spheroids with little spheroid settling or “flattening” of cell colonies in either static or suspension configurations. In BSA‐coated suspension systems, spheroid size scaled with the amount of BSA dissolved in culture medium. In static uncoated cultures, the normalized rat albumin production levels were enhanced by addition of BSA within culture medium. Thus, both addition of BSA to culture medium and application of BSA as a surface coating appear to be meaningful avenues for tailoring spheroid morphology and function. This 24‐well plate suspension culture system may be a valuable tool for high throughput investigations of liver cell behavior in a stable, uniform, 3D spheroid state. Biotechnol. Bioeng. 2013; 110:2548–2555. © 2013 Wiley Periodicals, Inc.

Aberrant connectivity is implicated in many neurological and psychiatric disorders, including Alzheimer’s disease and schizophrenia. However, other than a few disease-associated candidate genes, we know little about the degree to which genetics play a role in the brain networks; we know even less about specific genes that influence brain connections. Twin and family-based studies can generate estimates of overall genetic influences on a trait, but genome-wide association scans (GWASs) can screen the genome for specific variants influencing the brain or risk for disease. To identify the heritability of various brain connections, we scanned healthy young adult twins with high-field, high-angular resolution diffusion MRI. We adapted GWASs to screen the brain’s connectivity pattern, allowing us to discover genetic variants that affect the human brain’s wiring. The association of connectivity with the SPON1 variant at rs2618516 on chromosome 11 (11p15.2) reached connectome-wide, genome-wide significance after stringent statistical corrections were enforced, and it was replicated in an independent subsample. rs2618516 was shown to affect brain structure in an elderly population with varying degrees of dementia. Older people who carried the connectivity variant had significantly milder clinical dementia scores and lower risk of Alzheimer’s disease. As a posthoc analysis, we conducted GWASs on several organizational and topological network measures derived from the matrices to discover variants in and around genes associated with autism ( MACROD2 ), development ( NEDD4 ), and mental retardation ( UBE2A ) significantly associated with connectivity. Connectome-wide, genome-wide screening offers substantial promise to discover genes affecting brain connectivity and risk for brain diseases.

Aim: To identify ongoing practices and opportunities for improving national comparative effectiveness research (CER) translation through dissemination and implementation (D&I) via NIH-funded Clinical and Translational Science Award (CTSA) institutions. Materials & methods: Key informant interviews were conducted with 18 CTSA grantees sampled to represent a range of D&I efforts. Results & conclusions: The institutional representatives endorsed fostering CER translation nationally via the CTSA Consortium. However, five themes emerged from the interviews as barriers to CER D&I: lack of institutional awareness, insufficient capacity, lack of established D&I methods, confusion among stakeholders about what CER actually is and limited funding opportunities. Interviewees offered two key recommendations to improve CER translation: development of a centralized clearing house to facilitate the diffusion of CER D&I resources and methods across CTSA institutions; and formalization of the national CTSA network to leverage existing community engagement relationships and resources for the purpose of adapting and disseminating robust CER evidence locally with providers, patients and healthcare systems.

Objective— Matrix gla protein (MGP) inhibits arterial and cartilaginous calcification. A threonine to alanine (Thr83Ala) polymorphism (codon 83) in MGP is associated with myocardial infarction and femoral artery calcification. We examined the association of the MGP Thr83Ala polymorphism with quantity and progression of coronary artery calcification (CAC), a noninvasive measure of subclinical coronary atherosclerosis. Methods and Results— In 605 participants of the Epidemiology of Coronary Artery Calcification Study, generalized linear mixed models were fit to determine the association of MGP Thr83Ala with CAC quantity and progression. There was a significant additive relation between MGP Thr83Ala and CAC progression ( P =0.001). In the fully adjusted model, every 1 Ala83 allele increase was associated with an estimated 1.9% (95% confidence interval, 0.7%–3.0%) per year since baseline larger increase in CAC quantity. A proxy single nucleotide polymorphism for MGP Thr83Ala (rs6488724) was similarly associated with CAC progression in an independent cohort from the Genetic Epidemiology Network of Arteriopathy (GENOA) study. Conclusion— Increased risk of myocardial infarction associated with MGP ThrAla83 genotype observed elsewhere may be related to faster progression of subclinical coronary atherosclerosis. MGP genotype could be a potential candidate for identifying individuals at increased risk of atherosclerotic disease who would benefit from aggressive primary prevention strategies.

ABSTRACT Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.